Voltage-gated calcium channels are a diverse family of proteins which have a variety of biological functions, including presynaptic neurotransmitter release and protein signaling within the cell (Bito et al. (1997) Curr. Opin. Neurobiol. 7, 419-429; Dunlap et al. (1995) Trends Neurosci. 18, 89-98). The calcium currents produced by these channels are classified into P/Q-, N-, L-, R-, and T-type based on their pharmacological and biophysical properties, and all are expressed in brain (Dunlap et al. (1995) Trends Neurosci. 18, 89-98; Varadi et al. (1995) Trends Pharmacol. Sci. 16, 43-49; Nooney et al. (1997) Trends Pharmacol. Sci. 18, 363-371; Perez-Reyes et al. (1998) Nature 391, 896-900). Except for the T-type, whose molecular structure is unknown, all voltage-gated calcium channels are composed of at least three subunits, xcex11, xcex12xcex4 and xcex2 (De Waard et al. Structural and functional diversity of voltage-activated calcium channels. In Ion Channels, (ed. T. Narahashi) 41-87 (Plenum Press, New York, 1996)). A fourth subunit, xcex3, is associated with skeletal muscle calcium channels. The mRNA for this xcex3 subunit is abundant in skeletal muscle, but has not been detected in brain (Jay, S. D et al. (1990) Science 248, 490-492; Ludwig et al. (1997) J. Neurosci. 17, 1339-1349). Whether xcex3 subunits specific for brain calcium channels exist, remains to be determined. The xcex11 subunit forms the membrane pore and voltage-sensor and is a major determinant for current classification. Several isoforms of xcex11, arising from different genes, have been identified. The other subunits modulate the voltage-dependence and kinetics of activation and inactivation, and the current amplitude (Walker et al. (1998) Trends Neurosci. 21, 148-154). There is currently only one known gene and isoform for the xcex12xcex4 subunit, and four different xcex2 subunit genes encoding the distinct xcex2 isoforms. P/Q- and N-type channels purified from brain contain xcex11A and xcex11B subunits, respectively. These xcex11 subunits are associated with various proportions of the four separately encoded xcex2 subunit proteins, indicating that considerable subunit complexity exists (Scott et al. (1996) J. Biol. Chem. 271, 3207-3212; Liu et al. (1996) J. Biol. Chem. 271, 13804-13810).
A number of compounds useful in treating various diseases in animals, including humans, are thought to exert their beneficial effects by modulating functions of voltage-gated calcium channels. Many of these compounds bind to calcium channels and block, or reduce the rate of influx of calcium into cells in response to depolarization of the inside and outside of the cells. An understanding of the pharmacology of compounds that interact with calcium channels, and the ability to rationally design compounds that will interact with calcium channels to have desired therapeutic effects, depends upon the understanding of the structure of channel subunits and the genes that encode them. The identification and study of tissue specific subunits allows for the development of therapeutic compounds specific for pathologies of those tissues.
Cellular calcium homeostasis plays an essential part in the physiology of nerve cells. The intracellular calcium concentration is about 0.1 uM compared with 1 mM outside the nerve cell. This steep concentration gradient (xc3x9710,000) is regulated primarily by voltage-gated calcium channels. Several pathologies of the central nervous system involve damage to or inappropriate function of voltage-gated calcium channels. In cerebral ischaemia (stroke) the channels of neurons are kept in the open state by prolonged membrane depolarisations, producing a massive influx of calcium ions. This, in turn activates various calcium/calmodulin dependent cellular enzyme systems, e.g. kinases, proteases and phospholipases. Such prolonged activation leads to irreversible damage to nerve cells.
Certain diseases, such as Lambert-Eaton Syndrome, involve autoimmune interactions with calcium channels. The availability of the calcium channel subunits makes possible immunoassays for the diagnosis of such diseases. An understanding of them at the molecular level will lead to effective methods of treatment.
Epilepsies are a heterogeneous group of disorders characterized by recurrent spontaneous seizures affecting 1% of the population. In recent years several human genes have been identified, including the most recently identified potassium channels KCNQ2 and KCNQ3 for benign familial neonatal convulsions (Charlier et al. (1998) Nature Genet. 18, 53-55; Singh et al. (1998) Nature Genet. 18, 25-29; Biervert et al. (1998) Science 279, 403-406). To date, the involvement of voltage-gated calcium channels in epilepsies has been poorly characterized.
A number of mouse mutants have generalized tonic-clonic seizures, mostly resulting from gene knockouts. Ion channels are involved in many of these cases, including potassium (Smart et al. (1998) Neuron 20, 809-819), GABA (Homanics et al. (1997) Proc. Natl. Acad. Sci. USA 94, 4143-4148) and glutamate receptor channels (Brusa, et al. (1995) Science 270, 1677-1680). Comparatively fewer mouse models have been described with absence seizures, (also known as petit-mal or spike-wave), although this may be due to ascertainment bias as these seizures are associated with only a brief loss of consciousness. It has thus required a systematic electrocorticographic screen of known mutants to uncover mouse absence models. The mouse mutants ducky, lethargic, mocha, slow-wave epilepsy, stargazer and tottering, each show some form of spike-wave discharge associated with behavioral arrest which is characteristic of absence epilepsy (Noebels, J. L. In Basic Mechanisms of the Epilepsies: Molecular and Cellular Approaches., (ed. A. V. Delgado-Escueta, A. A. Ward, D. M. Woodbury and R. J. Porter) 44, 97-113 (Raven Press, New York, 1986); Noebels et al. (1990) Epilepsy Res. 7, 129-135; Cox et al. (1997) Cell 91, 1-20). The underlying genes are described in most of these models, and in twoxe2x80x94tottering and lethargicxe2x80x94the defect is in a gene encoding a neuronal calcium channel subunit (Fletcher et al. (1996) Cell 87, 607-617; Burgess et al. (1997) Cell 88, 385-392).
Because of the overlap in expression of voltage-gated calcium channel subunits and a limited understanding of tissue differences, it has not been straightforward to study the specific function of neuronal channels in vivo. The study of mouse mutations has begun to allow a dissection of this problem. For example, the neurological mutants tottering and lethargic have defects in genes encoding xcex11A and xcex24 subunits, respectively (Fletcher et al. (1996) Cell 87, 607-617; Burgess et al. (1997) Cell 88, 385-392). Their phenotypes are very similar, each exhibiting spike-wave seizures and moderate cerebellar ataxia without obvious neuronal damage. The nature of the mutation in each is commensurate with the respective roles of major and auxiliary calcium channel subunits: tottering has an amino acid substitution in the structural xcex11A subunit, whereas lethargic is not likely to express any functional xcex24 protein. The phenotype of the lethargic mouse shows that defects in regulatory subunits can also lead to the same neuronal malfunctions as observed for structural subunit mutations. Continued study of these mouse mutants will give further insight into neuronal calcium channel function in vivo.
The stargazer mutation arose spontaneously at The Jackson Laboratory on the A/J inbred mouse line (Noebels et al. (1990) Epilepsy Res. 7, 129-135), and was initially detected for its distinctive head-tossing and ataxic gait. Subsequent electrocorticography revealed recurrent spike-wave seizures when the animal was still, characteristic of absence epilepsy. The seizures were notably more prolonged and frequent than in tottering or lethargic mice, lasting on average six seconds and recurring over one hundred times an hour. The ataxia and head-tossing are presumed to be pleiotropic consequences of the mutation in the cerebellum and inner ear, respectively; the latter also distinguishes stargazer from the other mutants. The waggler mutant arose independently on the MRL/MpJ strain and was subsequently found to be an allele of stargazer (Sweet et al. (1991) Mouse Genome 89, 552). Waggler mice are severely ataxic but head-toss less frequently than stargazer and have a more pronounced side-to-side head motion.
The fine-mapping of the stargazer mutation on mouse chromosome 15 and the construction of a 1.3 Mb physical contig across the critical genetic interval was described in an earlier study (Letts et al. (1997) Genomics 43, 62-68). The present invention describes the first member of a multi-gene family that encodes a neuronal specific voltage-gated calcium channel xcex3 subunit disrupted in stargazer and waggler mice.
In one aspect, the present invention relates to a mammalian nucleic acid sequences encoding a neuronal-specific subunit of a voltage-gated calcium channel. Specifically disclosed are xcex32, xcex33 and xcex34 subunits. In other aspects, the invention relates to expression vectors which encode neuronal-specific subunits, as well as cells containing such vectors. In other aspects, the invention relates to antigenic fusion proteins comprising at least a portion of a mammalian neuronal-specific subunit of a voltage-gated calcium channel. Such fusion proteins are useful, for example, in the production of antibodies specifically reactive with the subunits of the invention. Such antibodies are also encompassed within the scope of the invention.
In another embodiment, the invention relates to a method for screening for compounds which modulate the activity of neuronal voltage-gated calcium channels. The method involves providing a cell transformed with a DNA expression vector comprising a mammalian cDNA sequence encoding a neuronal-specific xcex3 subunit of a voltage-gated calcium channel, the cell comprising additional calcium channel subunits necessary and sufficient for assembly of a functional voltage-gated calcium channel. The cell is contacted with a test compound and agonistic or antagonistic action of the test compound on the reconstituted calcium channels is determined.
Also encompassed within the scope of the invention are diagnostic methods based on the experiments described in the Exemplification section set forth below. These include, for example, a method of diagnosing Lambert-Eaton Syndrome and a method for diagnosing a defect in a xcex3 subunit gene of a patient with a neuronal disease such as epilepsy. Therapeutic methods based on the disclosure are also described.